This invention relates to DNA comprising a nucleotide sequence coding for at least a portion of a polypeptide which is a plant toxin of the ricin type, as hereinafter defined. It also relates to recombinant DNA molecules comprising a DNA sequence which codes for a polypeptide which is or is closely related to a natural plant toxin of the ricin type. Ricin, and also other plant toxins such as abrin, modeccin and viscumin, consist of two polypeptide chains (known as the A and B chains) linked by a disulphide bridge, one chain (the A chain) being primarily responsible for the cytotoxic and the other chain (the B chain) having sites enabling the molecule to bind to cell surfaces. Ricin is produced in the plant Ricinis communis (also known as the castor bean plant) via a precursor protein known as "preproricin".
Preproricin comprises a single polypeptide chain which includes a leader sequence. The leader sequence is subsequently removed in the organism to give proricin which is then cleaved to eliminate a linker region and joined by a disulphide bond to form the mature protein. The toxicity of ricin-type toxins operates in three phases: (1) binding to the cell surface via the B chain; (2) penetration of at least the A chain into the cytosol, and (3) inhibition of protein synthesis through the A chain attacking the 60S subunits of the ribosomes. Thus, separated A and B chains are essentially non-toxic, the inherently toxic A chain lacking the ability to bind to cell surfaces in the absence of the B chain.
It is also known that in ricin-type toxins the B chain binds to cell surfaces by virtue of galactose recognition sites, which react with glycoproteins or glycolipids exposed at the cell surface.
It has already been suggested that the toxicity of the ricin A chain might be exploited in anti-tumour therapy, by replacing the indiscriminately-binding B chain with a different carrier component having the ability to bind only to tumour cells. Thus, various immunotoxins have already been prepared, consisting of a conjugate of whole ricin or a separated natural ricin A chain and a tumour-specific monoclonal antibody. Although these known conjugates are of considerable potential in themselves, there is scope for improvement.
One problem with the known conjugates arises from a structural feature of the A chain from natural ricin. It is known that the natural ricin A chain becomes N-glycosylated during its synthesis, by enzymes present in Ricinus cells, and it is thought that the resulting sugar moieties are capable of non-specific interactions with cell surfaces. Thus, it appears that the known A chain conjugates are capable of a certain amount of binding with non target cells, even in the absence of the natural B chain, thus increasing the toxicity of such immunotoxins towards non target cells.
Another problem with the known ricin A chain conjugates stems from the fact that the B chain seems to have an important secondary function in that it somehow assists in the intoxication process, apart from its primary function in binding the ricin molecule to the cell surfaces. This secondary function is lost if the B chain is replaced by a different carrier component such as a monoclonal antibody.
If it were possible to prevent interactions between the cell surface via the A chain sugar moieties, whilst preserving the secondary toxicity-increasing function of the B chain, the toxicity of a whole ricin antibody conjugate towards normal cells could be reduced, and towards target cells could be increased, thus improving the therapeutic index of the immunotoxin. It is also known that the natural ricin B chain is N-glycosylated and the B chain sugar moieties may also contribute to non specific interactions, Also, the sugar moieties in both chains enable the ricin molecule to be sequestrated by reticuloendothelial cells in the liver, and so would lead to the rapid excretion from the system of a drug based on a part or the whole of the ricin molecule in which such sugar moieties were still present.
Attempts to remove all the sugar moieties from natural ricin by chemical or enzymatic methods have so far failed. Nevertheless the major obstacle confronting the use of known whole ricin-antibody conjugates is the presence of two galactose binding sites in the ricin B chain. These B chain galactose binding sites are primarily responsible for the non-specific cellular interactions of current whole ricin-antibody conjugates, particularly when used in vivo. Their presence in the natural toxin clearly eliminates or reduces the targeting specifity conferred by the antibody.
An improved immunotoxin based on ricin or another plant toxin of the ricin type, not suffering from these problems, could consist of a whole toxin molecule modified so that it is not N-glycosylated, and so that the B chain has no galactose recognition sites, but retains its secondary intoxication-promoting properties, coupled to a carrier moiety which delivers the toxin to the target cells. This could be a tumour-specific or cell/tissue specific vehicle such as a suitable monoclonal antibody.
Our research which has so far been concentrated on ricin itself, has indicated that the assembly of ricin (and the related agglutinin which consists of two ricin-like molecules with slightly modified A and B chains) does not involve the separate synthesis of the A and B chains as the products of distinct mRNA's, but rather the initial formation of a single polypeptide precursor containing both the A chain and B chain sequences. This is thought to apply in the case of other toxins of the same type.